We have shown that p19ARF protein binds and colocalizes with c-Myc protein, both exogenously and endogenously. ARF binding to c-Myc inhibits c-Myc transactivation of target genes and c- Myc-induced hyperproliferation and transformation. Furthermore, ARF does not inhibit repression of c-Myc target genes and actually appears necessary for Inr-mediated repression and enhances c- Myc-induced apoptosis. ARF also inhibits the proteolysis of c-Myc. The further characterization and functional relevance of this interaction is the focus of this proposal. Our hypothesis is that direct ARF interaction with c-Myc selectively regulates the activity of c-Myc protein. Therefore, loss of this important checkpoint control would contribute to deregulation of c-Myc function leading to hyperproliferation, transformation and inhibition of apoptosis. To test this hypothesis the following specific aims will be performed. Specific Aim I will be to characterize the biochemical interaction between c-Myc and ARF. Specific Aim 2 will be to determine the molecular mechanism that mediates the regulation of c-Myc activity by ARF. Specific Aim 3 will be to determine the biological role of the interaction of ARF with c-Myc. Our novel findings and the results of experiments proposed in these specific aims will have major implications for the function of c-Myc, ARF and p53. Our findings have already impacted the controversial model on the molecular function of c-Myc and will likely continue to impact the course of c-Myc studies on how c-Myc functions at the molecular level to elicit such powerful control over cellular proliferation, tumorigenesis and apoptosis. Finally, the inhibition of c-Myc-induced transformation and enhancement of c-Myc-induced apoptosis by ARF has direct therapeutic significance for cancer treatment.